This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is defective in patients with cystic fibrosis (CF). Most patients carry a copy of the gene that has lost a single amino acid at position 508 ([unreadable]F508-CFTR). The resulting protein folds inefficiently, but when rescued by chemical chaperones, exhibits some chloride-channel activity and thus may help to ameliorate disease symptoms. Unfortunately, the rescued protein is relatively unstable. We have shown that the CFTR-Associated Ligand CAL mediates post-maturational degradation of CFTR, and that inhibitors targeting the CAL PDZ binding domain stabilize [unreadable]F508-CFTR at the apical surface of airway epithelial cells. However, this effect requires specificity for the CAL PDZ domain vs. PDZ domains of other proteins with opposite effects on CFTR functional expression but overlapping specificity with CAL. We can assess in vitro binding of our inhibitors to individual candidate PDZ domains expressed recombinantly. However, we do not currently have an assay to determine the selectivity of this interaction within the cellular milieu nor with other potential off-target interactions. The goal of this pilot project is to develop and optimize methodologies for evaluating the efficacy and specificity of peptide and non-peptide CAL inhibitors in cell lysates, using pull-down approaches. The results will help us to evaluate candidate inhibitors as a basis for proof-of-principle for CFTR stabilization studies.